Cellular hypersensitivity to human pancreatic B-cell clone in diabetes mellitus and its relationship to the presence of islet cell antibodies.

A leukocyte migration inhibition test on the human pancreatic B-cell clone (JHPI-1) was performed in 13 IDDM patients with islet cell cytoplasmic antibody (ICCA) and/or islet cell surface antibody (ICSA), 15 IDDM patients without ICCA or ICSA, 34 NIDDM patients and 17 healthy controls. The mean values for the migration index (M.I. %) in each group were 85.4 +/- 6.9, 89.1 +/- 10.9, 98.3 +/- 7.9 and 100.0 +/- 8.5. The M.I. values were significantly decreased in IDDM patients than in NIDDM patients and controls irrespective of whether or not there were islet cell antibodies in the patients' sera. When M.I. values less than 0.83 (Mean-2 S.D.) were taken as indicative of inhibition, the percentage of IDDM and NIDDM patients with migration inhibition were 32% and 0% respectively. And the decreased M.I. values in IDDM patients proved not to be due to non-specific migration inhibition by normal M.I. values, with the human fetal lung fibroblast cells (W 138) as antigen. Our data suggested that the lymphocytes of IDDM patients might be sensitized by pancreatic B-cell antigen(s) present in the JHPI-1 cells, which promoted leukocyte migration inhibition. No correlation between the migration indices and duration of diabetes mellitus in IDDM patients was observed (r = 0.254, Y = 84.9 + 0.49 X). LMT to JHPI-1 seems to be useful in detecting the abnormal cell-mediated immunity even in patients with longstanding IDDM.

[The effect of methyleneblue on insulin and glucagon release stimulated by glucose and arginine in the isolated perfused rat pancreas].

To examine the role of NADPH in the release of insulin and glucagon, isolated rat pancreata were perfused with methyleneblue, which is known to oxidize NADPH. Hormonal release was stimulated by changes in arginine or glucose concentrations as follows. After establishing the basal secretion state during perfusion at various glucose levels for 10 min., pancreata were stimulated by the addition of arginine or a change in glucose concentration of the perfusate for 15 min. Conditions for the stimulation were: (A) addition of 10 mM arginine at constant 4 mM glucose concentration; (B) increase in glucose concentration from 2.8 mM to 11.1 mM, or (C) decrease in glucose concentration from 11.1 mM to 2.8 mM. In some experiments, methyleneblue was added throughout the perfusion period at 1 or 3 micrograms/ml. The effluent from the portal vein was collected over 1 minute intervals: Insulin and glucagon concentrations in the effluent were determined by radioimmunoassay. Insulin release. Stimulation by the addition of arginine and increased glucose concentration produced a typical biphasic insulin response. In both cases, 1 microgram/ml methyleneblue reduced the second phase, and 3 micrograms/ml methyleneblue inhibited both phases almost completely. Glucagon release: Stimulation by arginine and inhibition by increasing glucose concentration were not influenced by methyleneblue; however, glucagon release induced by lowering of glucose concentration was suppressed by 3 micrograms/ml of methyleneblue. Thus, methyleneblue specifically inhibits glucose- and arginine-induced insulin release while it has no effect on arginine-induced glucagon release.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical usefulness of artificial endocrine pancreas "Biostator" in management of unstable diabetics.

The diurnal blood glucose profiles of 15 unstable diabetics were continuously monitored by the artificial endocrine pancreas (AEP, Biostator). Then, feedback control (FC) with the AEP was performed on each subject for 24-32 hr. Based on the data obtained from FC, the patients received an intensified conventional insulin therapy (ICIT) consisting of two daily mixed injections, 45-60 min prior to meals. There was a negative correlation between the MAGE and the sum of serum CPR in a 50 g OGTT, before FC. The M-value, MBG and MAGE decreased significantly during FC and remained lower one month later, compared with those values obtained before FC. The ICIT caused a noticeable decrease in HbA1 levels one to four months after FC. The ICIT was characterized by an increase in the proportion of short-acting insulin in the daily insulin dosage to 40. 3 +/- 11.5%. The proportion of daily insulin dosage to body weight also increased to 0.73 +/- 0.17 U/kg. These results suggest that the AEP, Biostator, could be useful in the clinical management of unstable diabetics by providing a more precise estimate of patients insulin requirements, especially those of short-acting insulin, leading to a better long-term control of blood glucose.

[Hyperglycemia and inhibition of glucose-induced insulin release in 6-aminonicotinamide treated rats].

The effects of 6-aminonicotinamide (6-AN) on blood glucose and insulin release were studied in rats. 6-AN at 4mg/100g body weight slowly raised blood glucose concentrations to a significantly higher level than the control values 6 hours after an intraperitoneal injection. At this time severe glucose intolerance and low IRI response were noticed during an intravenous glucose tolerance test. Adrenalectomized rats replaced by hydrocortisone also presented hyperglycemia, glucose intolerance and low IRI response during IVGTT under treatment with 6-AN but to a lesser extent than in the intact rats. In in vitro experiments, decreased insulin release from the perfused pancreata of rats pretreated with 6-AN was found both in the 1st and 2nd phases of response to glucose stimulation. These data indicate that 6-AN-induced hyperglycemia is attributed to the inhibition of insulin release in adrenalectomized rats except for the hypothetical effect of 6-AN which diminishes an action of insulin on cellular glucose transport.

The effect of hypoxia on insulin and glucagon secretion in the perfused pancreas of the rat.

It has been reported that insulin secretion decreases during hypoxia both in vitro and in vitro, while an increase in glucagon secretion is found only in vivo. The effect of acute hypoxia on the secretion of glucagon and insulin was studied in the perfused rat pancreas. Phentolamine, an alpha-adrenergic blocker, was perfused during the period of hypoxia to elucidate the role of alpha-adrenergic stimulation. Sodium ATP and dibutyryl cAMP were also administered to study their effects on insulin and glucagon responses during hypoxia. In the present experiments, insulin secretion was suppressed while glucagon secretion was increased during hypoxia. Phentolamine did not cause any change in insulin of glucagon secretion. When dibutyryl cAMP was added, the increased glucagon secretion was reduced to the basal level, whereas the decreases in insulin secretion were not altered. The addition of sodium ATP reversed the hypoxia-induced decrease in insulin and the increase in glucagon secretion. These results suggest that a decrease in ATP production, which leads to impaired cAMP generation, pays a role in, and that alpha-adrenergic stimulation does not participate in the changes in, insulin and glucagon secretion during hypoxia in vitro.

Human pancreatic islet cell clones secreting insulin, glucagon and somatostatin: immunocytochemical and functional studies.

Human pancreatic A-, B- and D-cell clonal strains, named JHPG-1, JHPI-1 and JHPS-1, were established successfully from adult and fetal pancreata by the single cell plating feeder layer method using a modified Rose's chamber. This is the first time that insulin-, glucagon- and somatostatin-producing clonal strains have been separated into continuous clonal cell lines. The cultured cells are epithelial in nature, free of fibroblast contamination, and can be cloned. Under the phase-contrast microscope, the B-cell clone (JHPI-1) was generally oval or round in shape, the A-cell clone (JHPG-1) was bipolar, and the D-cell clone (JHPS-1) was nerve-like with cytoplasmic processes. By the use of immunocytochemical techniques, insulin-, glucagon- and somatostatin-like immunoreactivities were detected in each clone respectively. By radioimmunoassay it was revealed that each clone produced a single pancreatic hormone. The B-cell clone especially, was found to secrete insulin amply and continuously, for over 150 days. The glucagon release responses by the A-cell clone to insulin and glucose were also studied. The clonal strains obtained in this study provide useful systems for the investigation of the cell-biological aspects of human islet cells in vitro.

Adrenergic modulation of insulin and glucagon secretion from the isolated perfused rat pancreas.

In order to observe the effect of the adrenergic system on pancreatic glucagon secretion in the isolated perfused rat pancreas, phenylephrine, an alpha-adrenergic agonist, and isoproterenol, a beta-adrenergic agonist, were added to the perfused solution. 1.2 microM phenylephrine suppressed glucagon secretion at 2.8 mM glucose, and it also decreased insulin secretion at 11.1 mM glucose. 240 nM isoproterenol enhanced glucagon secretion not only at 2.8 mM glucose, but also at 11.1 mM glucose, as well as insulin secretion at 11.1 mM. In order to study the role of intra-islet noradrenalin, phentolamine, an alpha-adrenergic antagonist, and propranolol, a beta-adrenergic antagonist, were infused with the perfused solution. 10 and 100 microM phentolamine caused an increase in insulin secretion, and 25 microM propranolol decreased insulin secretion, while they did not cause any change in glucagon secretion. From these results, it can be concluded that alpha-stimulation suppresses not only insulin but also glucagon secretion, while beta-stimulation stimulates glucagon secretion, as well as insulin secretion. Intra-islet catecholamine may have some effect on the B cell, whereas it seems to have no influence on the A cell.

Synthesis and release of proinsulin and insulin by isolated rat islets of Langerhans.

Isolated rat islets of Langerhans were incubated for 60, 120, and 180 min and the incorporation of leucine-(3)H into proinsulin and insulin moieties was followed. Synthesis and release of these hormones could be followed by separate extractions of islets and incubation media. RELEASE OF NEWLY SYNTHESIZED PROINSULIN AND INSULIN OCCURRED UNDER THE FOLLOWING CONDITIONS: (a) incubation for greater than 60 min; (b) glucose concentrations above 5.3 mmoles/liter; (c) incubation with 5 mM dibutyryl cyclic AMP or theophylline in 5.3 mM glucose (potentiated by 16 mM glucose); and (d) incubation with 5 mM tolbutamide and 16 mM glucose. Synthesis of proinsulin and insulin was enhanced by time of incubation, high glucose concentrations, by dibutyryl cyclic AMP or theophylline, and by tolbutamide only at 16 mM glucose. Synthesis was totally inhibited by tolbutamide at 5.3 mM glucose.

Studies on the biological activity of porcine proinsulin.

The biological activity of purified porcine proinsulin was investigated in rats. In vivo studies revealed that proinsulin produced a hypoglycemic response similar to insulin but of lesser magnitude. Hypophysectomized and adrenalectomized animals proved to be more sensitive to proinsulin than normal. In vitro studies with rat hemidiaphragm were consistent with the in vivo findings. No competition with insulin action could be demonstrated. Experiments were carried out to determine whether proinsulin is converted to intermediate forms or insulin as a requisite to its biological activity. Labeled proinsulin injected in vivo or incubated in vitro remained intact by a variety of techniques (Sephadex column chromatography and polyacrylamide-gel electrophoresis). An inhibitory action of Kunitz pancreatic trypsin inhibitor on proinsulin action in vitro was confirmed. No clarification of this effect could be ascertained.